ABSTRACT The limited efficacy of the available therapy for squamous cell carcinoma of the head and neck (SCCHN) has prompted us to design a novel effective combinatorial immunotherapy for this disease. In this strategy, T cells engineered with a tumor antigen (TA)-specific chimeric antigen receptor (CAR) are used as the effector mechanism, since this approach allows specificity of tumor recognition and self amplification due to T lymphocyte self renewal capacity. We have selected chondroitin sulfate proteoglycan 4 (CSPG4) as the target, since i) CSPG4 is highly expressed in about 60% of SCCHN tumors with limited heterogeneity within each tumor; ii) CSPG4 is expressed on both differentiated SCCHN cells and SCCHN cells with high aldehyde dehydrogenase activity. The latter cells referred to as ALDHbright cells, display the characteristics of cancer initiating cells (CICs), since they are drug-resistant, express stemness genes and are tumorigenic in immunodeficient mice. Therefore, CSPG4 CAR T cells target both differentiated SCCHN cells and SCCHN CICs. According to the cancer stem cell theory, CICs have to be completely eliminated for a therapy to be successful, since these cells play an important role in disease recurrence and in metastatic spread; and iii) CSPG4 is not detected in normal tissues except for activated pericytes in the tumor microenvironment. Therefore, immunotargeting of CSPG4 with CAR T cells is expected to inhibit neo-angiogenesis in the tumor microenvironment and to contribute to the elimination of SCCHN cells, even those with low or lack of CSPG4 expression. In recent studies, we have shown that CSPG4 CAR T cells are effective in eliminating CSPG4+ SCCHN cells in vitro under normoxic conditions. In addition, they significantly inhibit the growth of human SCCHN tumors in immunodeficient mice but they do not eradicate them. This proposal will test our working hypothesis that the hypoxia driven hostile microenvironment of SCCHN tumors causes CAR T cell dysfunction and reduces SCCHN cell susceptibility to CAR T cell mediated lysis. T cell plasticity allows us i) to restore CAR T cell function by disrupting PD-1/PD-L1 axis to counteract CAR T cell ?exhaustion? and ii) to enhance viability and anti-tumor activity of CAR T cells by selectively increasing IL-15 level in the tumor microenvironment through the use of fusion proteins generated by linking IL-15 to anti-B7-H3 monoclonal antibody (mAb) HEK5. In addition, the susceptibility to CAR T cell mediated lysis of SCCHN cells will be restored by modulating anti-apoptotic molecule expression level through the inhibition with the small molecule sonidegib of hypoxia induced activation of Sonic Hedgehog Homolog pathway. The experiments will be performed in vitro utilizing SCCHN cell lines and in immunodeficient mice orthotopically grafted with both SCCHN cell lines and PDXs (some of which have already been established). The resulting information will determine whether the combinatorial strategy we have developed is effective in counteracting the hypoxia-related escape mechanisms utilized by SCCHN cells to avoid recognition and destruction by CSPG4 CAR T cells.